Large combinatorial libraries of biopolymers are starting points for isolating new enzymes, binding motifs and other useful molecules. For example, current technologies can generate populations of nucleic acids with complexities on the order of 1015 molecules and then isolate and identify a single molecule with a desired activity. Random polypeptide populations have greater chemical diversity than do polynucleotides, making them an attractive alternative to nucleic acids. Current systems are limited in their ability to easily generate large complex libraries of polypeptides that are in a form that allows the isolation and identification of rare molecules with a desired activity.
Described herein are ribozymes which join a nucleic acid (RNA or DNA) to a polypeptide, the resulting products and uses for these products. In one embodiment of the invention, a ribozyme which joins RNA to a polypeptide is used to produce a diverse library (or collection) of encoded polypeptides, in which library members are polypeptide-engineered mRNA conjugates. The polypeptide-engineered mRNA conjugates comprise an engineered mRNA, which is described below, and the translation product of the engineered mRNA. Diverse libraries of encoded polypeptides of the present invention can be screened to identify and isolate library members which are conjugates in which the polypeptide exhibits desired characteristics or properties (e.g., binding to a molecule or compound of interest, enzymatic activity). In a second embodiment, a ribozyme which joins RNA to a polypeptide is used to produce polypeptides, each of which is linked to and, thus, tagged by, a specific nucleic acid. The resulting nucleic acid-tagged polypeptides comprise a polypeptide (which is to be tagged by a nucleic acid); a peptide substrate of the ribozyie used to join the nucleic acid to the polypeptide; ribozyme RNA and a nucleic acid tag. The presence of the nucleic acid tag is useful, for example, in detecting, isolating, separating, identifying or purifying the polypeptide to which it is linked and in changing the properties (e.g., solubility) of the polypeptides. An unlimited number of nucleic acid tags can be made, which overcomes a limitation of presently-available methods, which rely on a small repertoire of affinity tags, as well as sometimes incompatible incubation protocols for each tag. Such ribozymes, methods of using them, the resulting products and methods of identifying, detecting, isolating, separating, purifying and/or using these products are described in detail herein.
One embodiment of the present invention relates to the use of ribozymes described herein to produce a diverse library or collection of encoded polypeptides in which the members are polypeptide-engineered mRNA conjugates; engineered mRNA; methods of producing diverse libraries of encoded polypeptides; methods of identifying target members of the libraries which are polypeptide-engineered mRNA conjugates which exhibit a desired activity or characteristic; target members and the components (polypeptide fragment and engineered mRNA fragment) of target members; and isolated ribozymes which join an mRNA to the translation product of the mRNA.
Polypeptide-engineered mRNA conjugates of the present invention comprise an engineered mRNA and the translation product of the engineered mRNA. Engineered mRNA of the present invention is one component of the conjugates which make up the diverse libraries of encoded polypeptides and is used to produce the conjugates. Members of the diverse library are polypeptide-engineered mRNA conjugates produced by in vitro translation of engineered mRNA. The engineered mRNA comprises: (a) a ribozyme RNA which specifically covalently links to a peptide; (b) a coding region for the peptide, referred to as a peptide tag, with which the ribozyme RNA specifically covalently links; (c) a coding region for a diverse polypeptide; and (d) two PCR primer-binding sites. In one embodiment, the engineered mRNA comprises, in order from the 5xe2x80x2 to the 3xe2x80x2 end, a ribozyme segment which comprises a first PCR primer binding-site and a motif that interacts specifically with a peptide; a coding region for the peptide with which the motif interacts; a coding region for a diverse polypeptide and a second PCR primer-binding site.
The ribozyme RNA which specifically covalently links to a peptide is present in (is a component or segment of) a ribozyme which is either a contiguous sequence (the entire ribozyme is a contiguous sequence) or comprised of two noncontiguous components: one which comprises the ribozyme RNA which specifically covalently links to the peptide and one which comprises the remainder of the ribozyme sequence. In the latter case, the ribozyme RNA which covalently links to a peptide is of sufficient length and appropriate composition to covalently link to the peptide tag in the presence of the remainder of the ribozyme sequence, under the conditions used to produce the diverse libraries. The remaining ribozyme sequence is the ribozyme sequence which, in combination with the component which covalently links to the peptide tag, makes up the complete ribozyme. The ribozyme sequence or segment which covalently links to the peptide tag can be as short as one nucleotide in length and can be from any location (e.g., 5xe2x80x2 end, internal segment, 3xe2x80x2 end) in the ribozyme. In one embodiment, the ribozyme segment includes from one to about 18 nucleotides, such as from the first to about the 18th nucleotide (from the 5xe2x80x2 end) of a ribozyme. In further embodiments, the ribozyme segment is the first 13 to 18 nucleotides (from the 5xe2x80x2 end) of the ribozyme. (e.g., the first 13, 14, 15, 16, 17, or 18 nucleotides from the 5xe2x80x2 end). The other component is the remaining ribozyme sequence (the remainder of the ribozyme which is necessary to form a functional ribozyme.). The two ribozyme components interact with one another to form a functional (complete) ribozyme under the conditions used to produce diverse libraries of encoded polypeptides. The 5xe2x80x2 end of the ribozyme RNA optionally comprises three phosphate groups or an mRNA cap, such as a 7-methyl guanosine triphosphate. Optionally, the engineered mRNA further comprises an RNA linker between the ribozyme sequence and the tag coding region. Optionally, the engineered mRNA additionally comprises a coding region for a peptide linker; this coding region is positioned between the coding region for the peptide tag and the diverse coding region. Further, the engineered mRNA can include an optional ribosome stalling site, which is located between the coding region for the diverse polypeptide segment and the second PCR primer-binding site.
In one embodiment, the engineered mRNA comprises (in order from 5xe2x80x2 to 3xe2x80x2): 1) a ribozyme sequence (also referred to as which-comprises a first PCR primer-binding site and a motif that interacts specifically with a peptide; 2) a coding region for a peptide (referred to as a peptide tag), with which the ribozyme motif interacts specifically; 3) a coding region for a diverse polypeptide segment and 4) a second PCR primer-binding site. In addition, in this embodiment, the engineered mRNA can include one or more of the following optional components: 5xe2x80x2 phosphate groups; an RNA linker between the ribozyme sequence and the tag coding region: a coding region for a peptide linker (positioned between the tag coding region and the diverse coding region) and a ribosome stalling site which is located between the coding region for the diverse polypeptide segment and the second PCR primer-binding site.
In a specific embodiment, the motif that interacts specifically with a peptide is modified Bovine Immunodeficiency Virus-1 (BIV-1) TAR RNA and the peptide tag is one with which BIV-1 TAR RNA interacts specifically by virtue of the BIV-1 Tat sequence embedded within the tag, e.g., the Tat tag peptide 1, also referred to as the Tat tag (SEQ ID NO.: 1: MSYSGPRPRGTRGKGRRIRRGGK), or the Tat 2 tag peptide, also referred to as the Tat 2 tag (SEQ ID NO.: 2: MKYSGPRPRGTRGKGRRIRRGGK). In both the Tat tag peptide and the Tat 2 tag peptide, the underlined amino acids are a BIV-1 Tat peptide (SEQ ID NO.: 3). The sequence of the modified BWV-1 TAR RNA is: 5xe2x80x2 GGA CAG CUC CGA GCA UUC UCG UGU AGC U (SEQ ID NO.: 4).
Also the subject of this invention are isolated ribozymes or ribozyme portions which join an mRNA to a polypeptide, such as the translation product of the mRNA. Such ribozymes comprise a motif which specifically covalently links to a peptide, with the result that the ribozyme is joined specifically to the peptide. Ribozymes of the present invention can be a contiguous sequence or can be comprised of two noncontiguous components which interact with one another to form the complete ribozyme, under the conditions used to produce diverse libraries of encoded polypeptides. The two noncontiguous components are a ribozyme segment which specifically covalently links to a peptide and a ribozyme segment which comprises the remaining ribozyme. As a result, an mRNA which comprises such a ribozyme and a coding region for the peptide with which the ribozyme interacts will be joined to the translation product of the mRNA (through binding and then covalent linkage of the ribozyme RNA and the peptide). An example of a ribozyme that contains a motif that interacts specifically with a peptide (modified TAR RNA) and joins itself specifically to certain peptides (e.g., the Tat tag peptide or the Tat 2 tag peptide) is specifically described herein.
Members of the encoded polypeptide libraries, referred to as target members, which have desired characteristics (e.g., binding, enzymatic, antigenic characteristics) are also the subject of this invention, as are methods of identifying target members. Polypeptide fragments and ribonucleoprotein fragments of members of the libraries, particularly fragments of target members of the libraries, are further subjects of this invention.
The present invention provides a method and reagents for generating diverse libraries of encoded polypeptides and for identifying and isolating members of the libraries, particularly-members which exhibit rare characteristics and occur in small numbers.
A second embodiment of the present invention relates to the use of ribozymes to join a desired nucleic acid tag to a polypeptide to produce a polypeptide tagged with the desired (specific) nucleic acid. As described above, the ribozyme can comprise a motif which specifically covalently links to a peptide. The resulting polypeptide-nucleic acid tag conjugate is a subject of the present invention, as are its components, collections or libraries of such conjugates and methods of making and using the conjugates. In this embodiment, a ribozyme which joins a polypeptide to a nucleic acid is used as follows to produce polypeptide-nucleic acid tag conjugates: A polypeptide, also referred to as a polypeptide of interest (e.g., a polypeptide to be identified, detected, separated, isolated, purified, altered in characteristic(s)), which is tagged with the peptide substrate of the ribozyme used, is joined covalently, as described below, to the nucleic acid tag through the action of the ribozyme. The resulting polypeptide-nucleic acid tag conjugate comprises: the polypeptide, the peptide substrate of the ribozyme used, a ribozyme RNA and the nucleic acid tag. In one embodiment, a polypeptide-nucleic acid tag conjugate comprises a polypeptide-nucleic acid tag conjugate comprising: (a) a polypeptide of interest; (b) a peptide tag linked to the polypeptide of interest, wherein the peptide tag is the substrate of a ribozyme; (c) a ribozyme or a segment of a ribozyme for which the peptide tag of (b) is a substrate, wherein the ribozyme or ribozyme segment is covalently linked to the peptide tag; and (d) a nucleic acid tag, wherein the nucleic acid tag is linked to the ribozyme or ribozyme segment. In one embodiment, the peptide is linked to the 5xe2x80x2 terminus of the ribozyme RNA and the nucleic acid tag extends from the 3xe2x80x2 terminus of the ribozyme RNA. The peptide substrate of the ribozyme is referred to herein as the peptide tag. As a result of the specific interaction of the peptide tag on the polypeptide with the ribozyme for which the peptide tag is a substrate, the polypeptide component is covalently linked to the nucleic acid tag. This technique has several clear advantages over other methods of tagging proteins. For example, because the peptide tag and the RNA component of the ribozyme interact specifically with each other, the ribozyme tagging reaction can be performed in a mixture of biological molecules, such as in a crude cell lysate, as well as in vivo. In addition, because there is no limit on the number of different possible nucleic acid tags, an essentially unlimited number of different (uniquely or specifically) tagged polypeptides can be produced. Once produced, the tagged proteins can be mixed and then detected simultaneously, using, for example, hybridization to DNA arrays.
Another embodiment of the present invention is a method of producing a polypeptide-nucleic acid tag conjugate, comprising: (a) combining: (1) a polypeptide of interest which bears a peptide tag wherein the peptide tag is a substrate for ribozyme RNA; (2) ribozyme RNA, wherein the ribozyme RNA specifically covalently links to the peptide tag of (a)(1) in the presence of the remainder of the ribozyme and has linked thereto a nucleic acid tag; (3) the remainder of the ribozyme, thereby producing a combination; and (b) maintaining the combination under conditions appropriate for ribozyme RNA of (a)(2) to associate with ribozyme RNA of (a)(3), forming a functional ribozyme that specifically covalently links the peptide tag to ribozyme RNA of (a)(2), whereby the polypeptide-nucleic acid tag conjugate is produced.
Another embodiment of the present invention is a method of separating a polypeptide from a mixture of polypeptides, comprising: (a) tagging the polypeptide at its amino terminus with a peptide tag, thereby producing a polypeptide-peptide tag, wherein the peptide tag is a substrate for ribozyme RNA, thereby producing a mixture comprising the polypeptide-peptide tag; (b) combining the mixture produced in (a) with (1) ribozyme RNA which bears a nucleic acid tag and specifically covalently links to the peptide tag in the presence of the remainder of the ribozyme and (2) the remainder of the ribozyme, under conditions appropriate for ribozyme (b) (1) to specifically interact with its peptide substrate thereby forming a covalent link with the peptide and tagging the polypeptide with the nucleic acid tag, whereby a mixture comprising a polypeptide-nucleic acid tag conjugate is formed; (c) combining the mixture formed in (b) with a nucleic acid which is a binding partner for the nucleic acid tag of the polypeptide-nucleic acid tag conjugate, whereby the binding partner hybridizes with the nucleic acid tag of the conjugate, forming a polypeptide-nucleic acid tag conjugate with the binding partner bound thereto; and (d) separating the polypeptide-nucleic acid tag conjugate with the binding partner bound thereto from the product of (c), whereby the polypeptide is separated from the mixture of polypeptides. In one embodiment, the binding partner is bound to a solid surface.
The present invention also relates to a method of detecting a polypeptide of interest in a mixture, wherein the method comprises: (a) combining a mixture to be assessed for the presence of the polypeptide-nucleic acid tag conjugate with a binding partner for the nucleic acid tag, wherein the binding partner (1) is a nucleic acid sequence sufficiently complementary to the nucleic acid tag that the binding partner and the nucleic acid tag of the conjugate bind to one another and remain bound under the conditions used and (2) is bound to a solid surface and wherein the combining occurs under conditions appropriate for binding of the nucleic acid tag and the binding partner and (b) detecting whether binding of the nucleic acid tag and the binding partner occurred, wherein if binding is detected, the polypeptide of interest is detected. In one embodiment, the binding partner or the nucleic acid tag is labeled with a detectable moiety (e.g., a chemical moiety, radioactivity) and detection of binding is carried out by detecting the presence of the detectable moiety.